Electrophoretic interactive spectral methods and devices for the detection and/or characterization of biological particles

ABSTRACT

Methods for identifying a biological particle in a sample medium include generating an Electrophoretic Quasi-elastic Light Scattering (EQELS) spectrum for the biological particle in the sample medium. The EQELS spectrum is compared to a reference database comprising a plurality of spectra, and each of the plurality of spectra correspond to an EQELS spectrum for one of a plurality of known biological particles. The biological particle in the sample medium is identified from the comparison.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/568,128, filed May 4, 2004, the disclosure ofwhich is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention generally relates to methods and systems forspectroscopy, and more specifically, to electrophoretic interactivespectral techniques.

BACKGROUND OF THE INVENTION

The rapid recognition of the presence of a cell and/or microbe, theirspecific identification and characterization, the selection of agent(s)for therapeutic intervention, and the identification andcharacterization of specific binding pairs involve clear medical andsecurity needs. The rapid detection of microbes, pathologic orotherwise, in humans, animals or in other environmental locations, suchas water, air, or food, may be used to provide an early response, forexample, to contain a pathogen and potentially save lives. Due, in part,to the modern centralization of food and drinking supplies, the earlydetection of microbes in these supplies could improve the safety of foodand/or water. As another example, the early and accurate recognition ofthe presence of a bioterror agent (e.g., ricin, anthrax, ebola, etc.) ina public area could potentially save lives by triggering an earlyresponse.

However, current technology generally requires either time-intensiveculturing of bacteria or other more expensive assays such asimmunological assays (e.g., Enzyme-Linked Immunosorbent Assay “ELISA”).Such technology can be time and/or labor intensive and may beprohibitively expensive.

SUMMARY OF THE INVENTION

According to embodiments of the present invention, methods and systemsfor identifying a biological particle in a sample medium are provided.An Electrophoretic Quasi-elastic Light Scattering (EQELS) spectrum isgenerated for the biological particle in the sample medium. The EQELSspectrum is compared to a reference database comprising a plurality ofspectra, and each of the plurality of spectra corresponds to an EQELSspectrum for one of a plurality of known biological particles. Thebiological particle in the sample medium is identified from thecomparison. The biological particle can be a biological cell or amicrobe selected from the group consisting of viruses, bacteria, fungi,and protozoa.

In some embodiments, the sample medium can be modified and a secondEQELS spectrum can be generated for the biological particle in thesample medium after modifying the sample medium. The EQELS spectra canbe compared and the biological particle in the sample medium can becharacterized based on the comparison. For example, the sample mediumcan be modified by any of the following: (i) adding a binder for atarget biological particle to the sample medium; (ii) adding a solventto the sample medium; (iii) changing a pH of the sample medium; (iv)changing a temperature of the sample medium; (v) changing an ionicstrength of the sample medium; (vi) adding an agent for altering bindingof a target biological particle to the sample medium; and/or (vii)adding a complexation agent for a target biological particle to thesample medium. Binders can be added to the sample medium, includingantibodies, cells, microbes, ligands, proteins, peptides, nucleic acids,polysaccharides, lipids, lipoproteins, haptens, and/or pharmaceuticalcompounds.

In particular embodiments, the sample medium includes a fluid selectedfrom the group consisting of: blood, blood products, water,cerebrospinal fluid, ascites, pleural fluid, and synovial fluid. Thebiological particle can be identified by determining at least onecharacteristic of the biological particle, such as an electrophoreticmobility, a biological particle concentration, a cytostatic character, acytotoxic character, a swimming rate, a biological particle volume, asurface charge, a binding strength, a binding constant, a bindingprofile, a ratio of a swim rate to an electrophoretic mobility, adiffusion constant, a biological particle size, a ratio of a dimensionto an electrophoretic mobility, a structure, a gyration ratio, a bindingenergy, a binding specificity, a binding site mapping, and an enzymeactivation on a surface of the biological particle.

In some embodiments, the sample is modified by adding an antibody to thesample medium. The antibody is a specific binder to a predeterminedbiological particle. The step of characterizing the biological particleincludes determining if the biological particle in the sample medium isthe predetermined biological particle from the comparison of the firstEQELS spectrum and the second EQELS spectrum. As another example,modifying the sample medium can include adding a therapeutic agent tothe sample medium. An effectiveness of the therapeutic agent can beassessed based on the comparison of the first EQELS spectrum and thesecond EQELS spectrum. The comparison can be used to determine whetherthe therapeutic agent binds to a surface of the biological particle, todetermine a change in the swim rate of a microbe, and/or to determine abinding constant for the therapeutic agent.

In some embodiments, the reference database includes swim rates for theplurality of known microbes and a swim rate for the microbe in thesample medium can be determined to identify the microbe. In otherembodiments, a microbe can be identified by determining the ratio of theswim rate of the microbe to the electrophoretic mobility of the microbe.The microbe can be identified based on its swim rate.

Detecting an EQELS spectrum can include exposing the biologicalparticles in the sample medium to an electric field, impinging lightfrom a light source on the biological particles in the sample medium toproduce scattered light, detecting the scattered light, and detecting aDoppler shift in the scattered light compared to light from the lightsource.

In particular embodiments, the biological particle is collected using afiltration device. A gas and/or liquid can be filtered with a filter totrap the biological particle, and the filter can be flushed with a fluidto provide the sample medium. The biological particle can be collectedautomatically.

In further embodiments according to the present invention, methods fordetecting a presence or absence of a specific binding pair in a samplemedium are provided. The specific binding pair includes a first memberand a second member. A first EQELS spectrum of the sample medium isdetected. The sample medium includes the first member of the specificbinding pair. A specimen is added to the sample medium. A second EQELSspectrum of the sample medium is detected after adding the specimen tothe sample medium. The first EQELS spectrum and the second EQELSspectrum are compared, and the presence or absence of the second memberof the specific binding pair in the sample medium is detected from thecomparison. The first member of the specific binding pair can be abiological particle, such as a microbe or biological cell.

In some embodiments, a first electrophoretic mobility of the firstmember of the specific binding pair is determined from the first EQELSspectrum, and a second electrophoretic mobility of the first member ofthe specific binding pair is determined from the second EQELS spectrum.The first EQELS spectrum and the second EQELS spectrum are compared bycomparing the first electrophoretic mobility and the secondelectrophoretic mobility.

In particular embodiments, a binding constant for the specific bindingpair is determined. Whether the first member of the specific bindingpair is activated and/or whether the first member of the specificbinding pair experiences cell death can also be determined. The specimencan be a drug, and a therapeutic and/or toxic effect of the drug can bedetermined from the comparison of the first EQELS spectrum and thesecond EQELS spectrum.

According to further embodiments of the present invention, methods ofassessing a biological particle in a medium include detecting an EQELSspectrum of the biological particle in the sample medium. The EQELSspectrum is compared to a reference database comprising a plurality ofspectra. Each of the plurality of spectra correspond to an EQELSspectrum for one of a plurality of known particles. A characteristic ofthe biological particle is assessed based on the comparison. Forexample, a diseased cell can be detected. In particular embodiments, thesample medium is a specimen of blood.

According to still further embodiments of the invention, systems foridentifying a biological particle in a sample medium include an EQELSspectrometer comprising and EQELS controller configured to detect anEQELS spectrum for the biological particle in the sample medium. Thebiological particle can be a biological cell or a microbe. An EQELSanalyzer is in communication with the EQELS spectrometer. A referencedatabase is in communication with the EQELS analyzer. The referencedatabase includes a plurality of spectra, and each of the plurality ofspectra corresponding to an EQELS spectrum for one of a plurality ofknown biological particles. The EQELS analyzer is configured to comparethe EQELS spectrum for the biological particle in the sample medium fromthe EQELS spectrometer with the plurality of spectra from the referencedatabase and to identify the biological particle in the sample mediumfrom the comparison.

According to further embodiments of the invention, systems for detectinga presence or absence of a specific binding pair in a medium areprovided. The specific binding pair includes a first member and a secondmember. An EQELS spectrometer includes an EQELS controller. The EQELScontroller is configured to detect a first EQELS spectrum of the samplemedium. The sample medium includes the first member of the specificbinding pair. A sample modification system is in communication with theEQELS controller and configured to add a specimen to the sample medium.The EQELS controller is configured to detect a second EQELS spectrum ofthe sample medium from the EQELS spectrometer after the samplemodification system adds the specimen to the sample medium. An EQELSanalyzer is in communication with the EQELS spectrometer and isconfigured to compare the first EQELS spectrum and the second EQELSspectrum and to detect the presence or absence of the second member ofthe specific binding pair in the sample medium from the comparison.

According to further embodiments of the invention, systems of assessinga biological particle in a sample medium include an EQELS spectrometerconfigured to detect an EQELS spectrum of the biological particle in thesample medium. An EQELS analyzer includes a reference databasecomprising a plurality of EQELS spectra. Each of the plurality ofspectra correspond to an EQELS spectrum for one of a plurality of knownbiological particles. The EQELS analyzer is configured to compare theEQELS spectrum of the sample medium to the plurality of spectra in thereference database and to assess a characteristic of the biologicalparticle based on the comparison.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a block diagram of an Electrophoretic Quasi-elastic LightScattering (EQELS) spectrometer according to embodiments of the presentinvention.

FIG. 2 is a block diagram of a specimen acquisition system according toembodiments of the present invention.

FIG. 3 is a block diagram of a flow-through EQELS spectrometer accordingto embodiments of the present invention.

FIG. 4 is a block diagram of data processing systems according toembodiments of the present invention.

FIGS. 5-7 are flow charts illustrating operations according toembodiments of the present invention.

FIG. 8 is a graph illustrating the effect of rFVIIIa on the binding ofzymogen FIX to activated platelets in the presence of 4 mM calciumchloride. Platelets are activated with 0.2 NIH U/mL thrombin followed bycomplete inhibition of thrombin with PPACK. In the absence of rFVIIIa,the binding constant (Kd) for zymogen FIX is 7.9 nM compared to a Kd of5.8 nM when rFVIIIa is present.

FIG. 9 is a graph illustrating the effect of calcium and rFVIIIa on thebinding constant for activated FIX (FIXa). Platelets are activated with0.2 NIH U/mL thrombin followed by complete inhibition of thrombin withPPACK. In the absence of calcium, no change in the plateletelectrophoretic mobility occurs, indicating no binding of FIXa. In thepresence of mM calcium chloride, but no rFVIIIa, a binding constant of1.9 nM is calculated by fitting data to a single state binding model. Inthe presence of 3 nM (5 U/mL) rFVIIIa and at 0 nM of FIX a, the plateletmobility is increased as a result of rFVIIIa binding. As theconcentration of FIXa is increased, the mobility is further increased,which reflects FIX a binding. In the presence of rFVIIIa, a Kd of 0.569nM is calculated.

FIG. 10 is a graph illustrating that rFVIIa activates platelet-boundFIX. Platelets are activated with 0.2 NIH U/mL thrombin followed byPPACK inhibition of thrombin. Then rFVIIa at 10 nM, calcium at 4 mM, and10 nM FIX are added to the activated platelets. The platelet suspensionis incubated for 10 minutes and the binding profile for the FIX isdetermined. If no activation of FIX occurred, the binding curve and thebinding coefficient would be similar to that shown in FIG. 8. It wasfound that the absence of rFVIIIa included in the medium, a bindingcoefficient for zymogen FIX of 1.62 nM is measured, similar to thatshown in FIG. 9 for the binding of FIXa to activated platelets. When 3nM rFVIIIa is present in the medium, the FIX Kd is 0.84 nM, similar tothat for FIXa plus rFVIIIa shown in FIG. 8. Because the bindingcoefficients similar to these for FIXa binding and not zymogen FIGbinding are found, activation by FIX is indicated.

FIG. 11A is a graph of the time and concentration dependence of rFVIIaon the activation of zymogen FIX. Platelets were activated with thrombinfollowed by inhibition of thrombin with PPACK. The activated plateletswere then loaded with rFVIIa (0.075 nM, 1 nM, or 10 nM) and monitored asa function of time. The mobility at the initial time (zero) is themobility for platelets loaded with zymogen FIX. RFVIIa does not bind toactivated platelets in the rFVIIa range of 0.75 to 10 nM. Changes in theplatelet mobility result from FIX activation.

FIG. 11B is a graph illustrating first order rate constants that werecalculated for the conversion of zymogen FIX to activated FIXa byrFVIIa. Four concentrations of rFVIIa are shown: 0.75 nM, 1 nM, 10 nM,and 20 nM.

FIG. 12 is a graph illustrating the amount of FIX activated as afunction of time and at different concentrations of rFVIIa (between 0 nMand 49 nM) for a chromogenic assay to show activation of FIX by rFVIIain the absence of TF. The inset of the graph shows the rate constant forFIX activation that corresponds to a specific concentration of rFVIIa.

DETAILED DESCRIPTION OF THE INVENTION

The present invention now will be described more fully hereinafter withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown. This invention may, however, be embodied inmany different forms and should not be construed as limited to theembodiments set forth herein; rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art. Likenumbers refer to like elements throughout. Elements in the variousfigures are not drawn to scale and may be enlarged to show detail.

The terminology used in the description of the invention herein is forthe purpose of describing particular embodiments only and is notintended to be limiting of the invention. As used in the description ofthe invention and the appended claims, the singular forms “a”, “an” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise.

“Target” as used herein refers to any type of particle for whichdetection may be desired, including but not limited to biologicalparticles, e.g., whole cells, microbes, peptides, proteins, nucleicacids, polysaccharides, lipids, lipoproteins, etc.

“Binding pair” refers to a pair of particles, one of which may be atarget, which members of the binding pair specifically and selectivelybind to one another. Examples of suitable binding pairs include, but arenot limited to: cells and ligands; microbes and ligands; nucleic acidand nucleic acid; protein or peptide and nucleic acid; protein orpeptide and protein or peptide; antigens and antibodies; receptors andligands, haptens, or polysaccharides, complementary nucleic acids,pharmaceutical compounds, etc. Members of binding pairs are sometimesalso referred to as “binders” herein.

The term “microbe” as used herein refers to viruses, bacteria, fungi,and/or protozoa.

The term “cell” as used herein refers to any type of cell, includinghuman cells, animal cells (such as swine cells, rodent cells, caninecells, bovine cells, ovine cells and/or equestrian cells) cloned cells,plant cells, or the like. The cells may be blood cells, cultured cells,biopsied cells, or cells that are fixed with a preservative. The cellscan be nucleated, such as white blood cells or suspended endothelialcells, or non-nucleated, such as platelets or red blood cells.

The following abbreviations are used herein: Recombinant activatedcoagulation factor VIIa (rFVIIa), Wild-type coagulation factor VIIa(FVIIa) tissue factor (TF), Coagulation factor IX (FIX), Activatedcoagulation factor IX (FIXa), Coagulation factor VIII (FVIII),Coagulation factor X (FX), Coagulation factor XI (FXI), Activatedcoagulation factor XI (FXIa), binding constant (Kd), prostacyclin(PGI2), platelet rich plasma (PRP), electrophoretic mobility (μ), changein electrophoretic mobility (Δμ), Confidence interval (CI), Standarderror (SE).

While the methods and apparatus of the present invention are sometimesexplained with respect to analyte and receptor binding pairs herein forpurposes of clarity, it is to be understood that the methods andapparatus of the instant invention may be applied to other targets,probes, and other binders.

Although embodiments of the present invention are describe with respectto Electrophoretic Quasi-elastic Light Scattering (EQELS) spectroscopyit should be understood that other electrophoretic interaction spectraltechniques (i.e., techniques in which a biological particle in anelectrophoretic field interacts with an energetic medium to generate aspectrum) can be used, such as dynamic light scattering (DLS) and photoncorrelation spectroscopy (PCS). Although embodiments of the presentinvention are describe are with respect to an excitation light beam,other energetic media can be used, including electromagnetic energy,acoustic energy, ultrasonic energy, or other suitable energy media. Forexample, electromagnetic energy can be employed from any suitablespectral range, such as visible light, infrared, ultraviolet, and/orx-ray ranges. For example, actinic radiation having a wavelength fromabout 200 nm to about 700 nm can be used as an energetic medium forinteraction with a biological particle in an electrophoretic field.Visible light radiation can be used in light-scattering techniques,including elastic light scattering and quasi-elastic light scattering.Ultraviolet radiation can be used, for example, in capillaryelectrophoresis systems having an ultraviolet laser as an energy sourcefor ultraviolet radiation impinged on a biological particle in thecapillary flow stream. Thus, any suitable energy source andcorresponding energy medium can be used.

According to particular embodiments of the present invention,Electrophoretic Quasi-elastic Light Scattering (EQELS) spectroscopy isused to identify a microbe in a medium and/or to detect the presence orabsence of a specific binding pair in a medium. Electrophoreticquasi-elastic light scattering is a laser spectroscopy techniquegenerally used to study the electrophoretic mobility of particles in asample. An exemplary EQELS spectrometer 10 is illustrated in FIG. 1. Thespectrometer 10 includes a laser 14 that impinges a beam of light onto asample 20. The sample 20 is positioned between two electrodes 28 thatprovide an electric field to the sample 20. Charged particles in thesample 20 are induced to move due to the application of the electricfield. For example, the sample 20 can include a sample medium in which abiological particle of interest is in a solution or suspension. Forexample, the sample medium can include blood, blood products, water,cerebrospinal fluid, ascites, pleural fluid, and/or synovial fluid.Movement of the particles in the sample 20 is detected by quasi-elasticscattering from the generally coherent light provided by the laser 14.Some of the incident photons will encounter moving particles in thesample 20. When this encounter occurs, a small amount of energy from thephoton is given up, and consequently, the frequency of the scatteredlight is slightly reduced. This scattered light is detected by adetector 26.

As illustrated in FIG. 1, the spectrometer 10 is connected to aprocessor 12 that includes an EQELS signal analyzer 22. The processor 12receives signals from the spectrometer 10, which are analyzed by theEQELS signal analyzer 22. For example, the scattered light detected bythe detector 26 can be analyzed to determine the magnitude of the smallshift in frequency. This shift in frequency is proportional to the rateof movement of the particle in the sample 20 and is detected as aDoppler shift. The signal analyzer 22 can measure the Doppler shiftthrough a heterodyne technique in which unshifted light is mixed withthe scattered light to produce “beats”. This signal is measured as anautocorrelation function that can then be Fourier transformed to yield apower spectrum for interpretation.

In some embodiments, the EQELS spectrometer 10 can be used to detectand/or characterize biological particles, such as biological cellsand/or microbes.

In particular embodiments, the EQELS spectrometer 10 is used to detectan EQELS spectrum for a sample 20 that includes a biological particle ina sample medium. The EQELS spectrum is compared to a database of knownspectra, each of the known spectra corresponding to one of a pluralityof known biological particles. The biological particle in the medium isidentified from the comparison.

In other specific embodiments, the presence or absence of a specificbinding pair in a sample medium is detected by the EQELS spectrometer10. A first EQELS spectrum of a sample medium including one member(e.g., the target) of the specific binding pair is detected. A specimenis added to the sample medium and a subsequent EQELS spectrum isdetected after adding the specimen. The EQELS spectra before and afterthe addition of the specimen are compared, and the presence or absenceof the second member of the specific binding pair in the sample mediumis detected based on the comparison.

In further particular embodiments, a biological particle is assessed.The EQELS spectrometer 10 is used to detect an EQELS spectrum for asample 20 that includes a biological particle. The EQELS spectrum iscompared to one or more known spectra of known biological particles. Acharacteristic of the biological particle can be assessed, such asdiseases or abnormalities, including congenital, neoplastic or otherconditions. Various characteristics can be used to assess the biologicalparticle, including electrophoretic mobility, the concentration of thebiological particle, cytostatic character, cytotoxic character, swimmingrate, particle volume, surface charge, binding strength, bindingconstant, binding profile, a ratio of the swim rate to theelectrophoretic mobility ratio, diffusion constant, particle size, aratio of the dimension of the particle to the electrophoretic mobility,structure factors, gyration ratio, binding energy, binding specificity,binding site mapping and/or enzyme activation on a surface of thebiological particle.

FIG. 2 is a block diagram of a sample acquisition system 30 according toembodiments of the present invention. The acquisition system 30 includesan acquisition chamber 36 that includes a filter 34, inlets 32 and 42and outlets 38 and 40. Valves (not shown) can be used to control flowbetween the inlets 32 and 42 or the outlets 38 and 40 and the chamber36. In the configuration shown in FIG. 2, a vacuum can be provided inoutlet 40 to create negative pressure in the chamber 36 so that testfluid enters the chamber 36 from the inlet 32. The test fluid can be agas or liquid, such as air or water. The test fluid then passes throughthe filter 34, and microbes and/or cells are filtered from the testfluid. After a specimen is collected on the filter 34, a solvent entersthe chamber 36 through the inlet 42. The solvent can combine withmicrobes and/or cells that have been collected on the filter to form amedium. The medium then exits the chamber through the outlet 38 to acollection area for subsequent EQELS spectroscopy or directly to anEQELS spectrometer. Although two inlets 32 and 42 and two outlets 38 and40 are shown in FIG. 2, it should be understood that otherconfigurations can be used to accomplish the functions described herein.For example, the outlet 40 and the inlet 42 can be combined to provide asingle inlet/outlet.

The acquisition system 30 in FIG. 2 can be used to automatically collecta sample for analysis from a fluid. For example, the acquisition system30 could be miniaturized, automated and/or combined with an EQELSspectrometer and placed in various locations to monitor an air, water,and/or food supply. The acquisition system 30 can be used for bioterrorsurveillance to collect samples of fluids in an environment and/ormonitor the collected samples for microbial agents. A telecommunicationssystem can also be provided to communicate the results of the EQELSspectra obtained. When a EQELS spectrum is obtained that indicates thepresence of a particular microbe is in the sample, a central command canbe alerted through the telecommunications system and/or an alarm can beactivated.

The acquisitions system 30 can also be used to add various antibodies tothe collected sample. For example, a pre-selected antibody withantigenic specificity against pathogens of bioterror significance couldbe added to a medium including the suspected microbe in the chamber 36,e.g., through the inlet 42. If the suspected microbe were present in thesample, the antibody may selectively modify the microbe's mobility. Thechange in mobility can be detected by a change in the EQELS spectraobtained before and after the addition of the antibody.

In particular embodiments according to the present invention, aflow-through device 50 can be provided as illustrated in FIG. 3. Theflow-through device 50 includes an inlet 54 and outlet 56 and a sampleregion 52 therebetween. The inlet 54 can include a valve (not shown) forcontrolling the flow of a sample medium into the sample region 52.Electrodes 58 are positioned on opposite sides of the sample region 52to produce an electric field. A light source 60 (S) impinges a lightbeam on the sampling region. The resulting scattered light is thendetected by a detector 62 (D).

As illustrated in FIG. 3, a sample medium including a microbe and/orcell of interest can flow into the sample region 52 through the inlet54. The inlet 54 can be closed, for example, using a valve (not shown),when a suitable amount of sample medium has entered the sample region52. The electrodes 58 can produce an electric field in the sample region52, and an EQELS spectrum can be obtained using the incident light fromthe light source 60 and scattered light from the detector 62. The samplemedium can then exit the sample region 52 through the outlet 56, forexample, using a fluid pump, suction mechanism, and/or other techniquesto remove fluid from a chamber including techniques known to those ofskill in the art. The outlet 56 can also include a valve (not shown) forcontrolling and directing fluid flow from the sample region 52. Anothersample medium can then flow through the inlet 54 for subsequent testing.In this configuration, several sample mediums can be tested in rapidsuccession. In some embodiments, the flow-through device 50 can beconnected to the acquisition system 30 in FIG. 2. It should beunderstood that other configurations of flow-through devices can be usedto perform operations according to embodiments of the present invention.For example, the inlet 54 and the outlet 56 can be replaced with asingle opening to provide a combined inlet/outlet for batch-typeoperation.

FIG. 4 is a block diagram of exemplary embodiments of data processingsystems that illustrates systems, methods, and computer program productsin accordance with embodiments of the present invention. A dataprocessing system 110 is provided that includes a processor 120 incommunication with an EQELS spectrometer 125, and a memory 114.Exemplary EQELS systems that can be used for the EQELS system 125 areillustrated in FIGS. 1 and 3. As illustrated in FIG. 4, the EQELS system125 includes an acquisition system 130 and a sample modification system135. The sample modification system 135 is configured to modify thesample in the spectrometer, such as by adding a substance, such as anantibody or a therapeutic agent, to the sample. An exemplary acquisitionsystem for acquiring a specimen for EQELS spectrometry is illustrated inFIG. 4. In some embodiments, the EQELS spectrometer 125, the samplemodification system 135 and/or the acquisition system 130 are omitted.For example, a sample can be positioned in an EQELS system 125 manuallywithout requiring a separate acquisition system 130 and/or spectra canbe obtained according to embodiments of the invention without modifyingthe sample with the sample modification system 135. In some embodiments,the EQELS spectrometer 125 is omitted and an EQELS spectrum obtainedfrom a remote EQELS spectrometer is provided to the data processingsystem 110 for analysis.

The sample modification system 135 can modify the sample, for example,by adding a binder for a target biological particle, adding a solvent,changing the pH of the sample medium, changing the temperature of thesample medium, changing the ionic strength of the sample medium, addingan agent for altering the binding characteristics of a target biologicalparticle, and/or adding a complexation agent for a target biologicalparticle. Examples of binders, include antibodies, cells, microbes,ligands, proteins, peptides, nucleic acids, polysaccharides, lipids,lipoproteins, haptens, and pharmaceutical compounds.

The processor 120 communicates with the memory 114 via an address/databus 148. The processor 120 can be any commercially available or custommicroprocessor. The memory 114 is representative of the overallhierarchy of memory devices containing the software and data used toimplement the functionality of the data processing system 110. Thememory 114 can include, but is not limited to, the following types ofdevices: cache, ROM, PROM, EPROM, EEPROM, flash memory, SRAM, and DRAM.

As shown in FIG. 4, the memory 114 may include several categories ofsoftware and data used in the data processing system 110: the operatingsystem 152; the application programs 154; the input/output (I/O) devicedrivers 158 and the data 156. The data 156 may include a database ofknown EQELS profiles 144 and/or collected EQELS data 146 from the EQELSsystem 125.

As will be appreciated by those of skill in the art, the operatingsystem 152 may be any operating system suitable for use with a dataprocessing system, such as OS/2, AIX, OS/390 or System390 fromInternational Business Machines Corporation, Armonk, N.Y., Windows CE,Windows NT, Windows95, Windows98, Windows2000, or WindowsXP fromMicrosoft Corporation, Redmond, Wash., Unix or Linux or FreeBSD, Palm OSfrom Palm, Inc., Mac OS from Apple Computer, LabView or proprietaryoperating systems. The I/O device drivers 158 typically include softwareroutines accessed through the operating system 152 by the applicationprograms 154 to communicate with devices such as I/O data port(s), datastorage 156 and certain components of the memory 114 and/or the EQELSspectrometer 125. The application programs 154 are illustrative of theprograms that implement the various features of the data processingsystem 110 and preferably include at least one application whichsupports operations according to embodiments of the present invention.The data 156 represents the static and dynamic data used by theapplication programs 154, the operating system 152, the I/O devicedrivers 158, and other software programs that may reside in the memory114.

While the present invention is illustrated, for example, with referenceto the EQELS profile analysis module 160 being an application program inFIG. 4, as will be appreciated by those of skill in the art, otherconfigurations may also be utilized while still benefiting from theteachings of the present invention. For example, the EQELS profileanalysis module 160 may also be incorporated into the operating system152, the I/O device drivers 158 or other such logical division of thedata processing system 110. Thus, the present invention should not beconstrued as limited to the configuration of FIG. 4, which is intendedto encompass any configuration capable of carrying out the operationsdescribed herein.

The I/O data port can be used to transfer information between the dataprocessing system 110 and the EQELS spectrometer 125 or another computersystem or a network (e.g., the Internet) or to other devices controlledby the processor. These components may be conventional components suchas those used in many conventional data processing systems that may beconfigured in accordance with the present invention to operate asdescribed herein.

Particular embodiments of the present invention can be used to detectbioterror agents in an environment that may be susceptible to thepresence or incursion of such agents, such as air, water, or foodsupplies, or in human or animal tissue. A sample of material from theenvironment of interest can be used to obtain an EQELS spectrum, and thebioterror agent can be identified and/or characterized by comparing theEQELS spectrum to a reference database of EQELS spectra of candidateagents to determine if the environment contains any of the candidateagents.

Embodiments according to the present invention will now be describedwith respect to the following non-limiting examples:

Example 1 Microbe Detection

As shown in FIG. 5, an EQELS spectrometer (such as the EQELSspectrometer 10 in FIG. 1) can be used to detect an EQELS spectrum for asample that includes a microbe in a medium (Block 200). The EQELSspectrum may be compared to a database of known spectra such that eachof the known spectra corresponds to one of a plurality of known microbes(Block 202). The microbe in the medium can be identified from thecomparison (Block 204). Microbes include viral, bacterial, fungal andprotozoa microbes, and in particular embodiments, cytomegolovirus (CMV),herpes simplex virus (HSV), Epstein-Barr virus (HBV), respiratorysyncytial virus (RSV) and human immunodeficiency virus (HIV).

For example, the EQELS spectrum can be used to determine theelectrophoretic mobility of a microbe, and the electrophoretic mobilitycan be used to identify the microbe. The electrophoretic mobility maydepend on the surface charge of the microbe and/or on frictional forcesresulting from the shape/size of the microbe and/or on the viscosity ofthe solvent. The surface charge on the microbe surface may also dependon solvent conditions, such as pH.

In some embodiments, the concentration of a microbe can be determined.The EQELS spectrum from a sample with an unknown microbial concentrationcan be compared with a spectrum from a sample with a knownconcentration. The integration of the spectrum (i.e., thearea-under-the-curve) can be used to determine the concentration.

The proof of the microbe's identity may be enhanced by the addition ofan antibody that binds to a specific microbe. When the antibody binds tothe microbe, it can change both the surface charge and/or the frictionalforces and thus, the antibody can change the electrophoretic mobility ofthe microbe. The electrophoretic mobility can be determined from theEQELS spectrum.

Embodiments of the present invention can be used to identify a microbe,for example, used for bioterror. A specific list of potential microbepathogens could be developed. If an initial screen of the sample showedthe presence of a microbe, a cocktail of antibodies that includedantibodies against microbes-of-interest would be added to the sample. Ifany change in the profile of the original mobility is observed, then thepresence of a microbe could be identified.

The sensitivity of a specific antibiotic or anti-microbial agent againsta specific microbe can also be determined. Without wishing to be boundby any particular theory, before an antibiotic or anti-microbial agentcan exert its therapeutic effect, it must first bind to the surface ofthe microbe. When the antibiotic or anti-microbial agent binds to thesurface, it can change the microbe surface charge and/or frictionalforces. An EQELS spectrum or spectra can be used to detect the changesin surface charge and/or frictional forces. Moreover, if the antibioticor anti-microbial agent is effective in producing either a cytostaticeffect or in killing the microbe, an effect on the swimming rate of themicrobe, the surface charge, and/or the microbe's volume (e.g., fromswelling) may be observed. Thus, EQELS spectra can be used to determinewhether an antibiotic or anti-microbial agent binds to a microbe and/orkills the microbe. This information may be useful to test a microbialsample for sensitivity to a particular antibiotic or anti-microbialagent.

The binding constant for an anti-microbial agent can also be determinedfrom an EQELS spectrum of a sample including the microbe and theanti-microbial agent. The binding constant can be used as an indicationof the effectiveness of therapy for the anti-microbial agent. Forexample, the concentration of the anti-microbial agent can be increasedover time in the microbial sample medium. Changes in the mobility of themicrobe as a function of the therapeutic agent can then be determinedfrom the EQELS spectrum. The resulting binding profile can be fitted toa binding model, such as a one-state-binding model and/or a higher statebinding model, to provide a binding constant.

Parameters that can be used to identify microbes and/or to assess theeffectiveness of an anti-microbial agent include swim rate (e.g., asdetermined by laser velocimetry), the ratio of the microbe swim rate tothe electrophoretic mobility, the diffusion constant, the dimensions ofthe microbe (e.g., as determined by the diffusion constant and/orincluding ratios of gyration, volume, characteristic dimension,structure factors, rod/cocci/axial ratios, etc.), and/or the ratio of amicrobe dimension (e.g., the largest dimension) and the electrophoreticmobility.

Examples of fluids for which EQELS spectrum can be obtained and variousmicrobes in the sample assessed include blood, blood products, water,air, cerebrospinal fluid, ascites, pleural fluid, synovial fluid and thelike.

Example 2 Cellular-Based Assays and Cellular Monitoring

In some embodiments, the presence or absence of a specific binding pairin a medium is detected by an EQELS spectrometer (such as the EQELSspectrometer 10 in FIG. 1). As illustrated in FIG. 6, an initial EQELSspectrum of a medium including one member of the specific binding pair(e.g., a cell) is detected (Block 210). A specimen is added to themedium (Block 212) and a subsequent EQELS spectrum is detected afteradding the specimen (Block 214). The EQELS spectra before and after theaddition of the specimen are compared (Block 216), and the presence orabsence of the other member of the specific binding pair in the mediumis detected based on the comparison (Block 218). The other member of thespecific binding pair can include any ligand that binds to a cellsurface, including chemical or biologic drugs and/or naturally occurringor man-made substances, such as growth factor, hormones, lymphokines,chemokines, lipids, antibodies, biochemicals and the like. A change inthe measured cell electrophoretic mobility can be detected based on theEQELS spectra before and after the addition of the specimen whenspecific binding occurs.

In other embodiments, a cellular specimen is assessed. As illustrated inFIG. 7, an EQELS spectrometer, such as the EQELS spectrometer 10 in FIG.1, is used to detect an EQELS spectrum for a sample that includes acellular specimen (Block 230). The EQELS spectrum is compared to one ormore known spectra of known cells (Block 232). A characteristic of thecellular specimen can be assessed (Block 234), such as diseases orabnormalities, including congenital, neoplastic or other conditions.

Differences in electrophoretic mobility detected by EQELS spectrometrycan be used to detect abnormal cells, normal cell binding therapeuticsor an abnormal ligand, and/or to provide detailed thermodynamic,biologic, clinical, and chemical information concerning cellularinteraction. Examples of characteristics that can be assessed includebinding constants, binding energies, binding specificity, and/or mappingof binding cites. For example, EQELS spectra can detect a change in theelectrophoretic mobility of a cell that is induced by ligand binding.The ligand binding constant can be obtained from the dependence of thechange in the cell electrophoretic mobility on the concentration of theligand. The binding constant is a useful property of the ligand-cellinteraction that can be related to the biological efficacy of theligand, mapping of a binding site, and/or the selection of a therapy.

For example, cells derived from a specific developmental cell line canexpress different surface epitopes. These differences can contribute tothe identification of a cell as a lymphocyte, granulocyte, T-cell,B-cell etc. This cell surface property results in different EQELSspectra that can be used to differentiate between leukemic blasts andnormal blood cells, between platelets and red blood cells, etc.Embodiments of the present invention can detect these differenceswithout using fixed (preserved) cells, incubation with a fluorescentlylabeled antibody, and/or a flow cytometry technique.

In some cases, ligand binding can lead to cell activation, such as withthrombin (or other platelet agonists) binding by platelets andFmetLuePhe binding by neutrophilic granulocytes. Another example of cellactivation is the activation of leukocytes. When a cell is activated,its surface changes to expose a different array of biologic molecules.These changes can result in a measurable difference in the cell surfacecharge and, hence, its electrophoretic mobility. When a cell dies,similar events can occur, including the loss of electrochemicalgradients across the cell membrane. These events may be detected bychanges in the EQELS spectra.

Without wishing to be bound by any particular theory, each type oftissue includes cells that has unique surface characteristics. Theuniqueness of the cell surface results from the expression of themolecular species on the cell's surface that permits the unique functionand ability for each cell line. If a given cell binds a ligand to itssurface or if the cell line becomes diseased, either through acongenital disease or an acquired disease, its cell surface changes. Achange in the cell surface can lead to changes in the cell's surfacecharges. An EQELS spectrum can be used to detect a change in the cellsurface charge. The EQELS spectrum can also be used to detect specificdrug binding, to detect the activation of enzymes on the cell surface,to distinguish normal cells from abnormal cells, to distinguish restingcells from activated cells, and/or to monitor drug efficacy and safety.

For any cellular therapeutic to be effective, it must first bind to atargeted cell surface. Therapeutic agents can include any ligand ordrug. The avidity or strength with which a therapeutic agent binds tothe cell often determines its usefulness or efficacy as a therapeuticagent. The interaction between a therapeutic agent and the cell(s) canbe assessed. The information that can be obtained from an EQELS spectrumincludes, without limitation, the nature of the biologic interaction,the chemical interaction, and/or the thermodynamic interaction betweenthe therapeutic agent and a targeted cell surface. In addition to thedetermination of cellular binding of the therapeutic agent, the bindingconstant and factors that effect binding, such as the concentration ofthe agent, temperature, pH, ionic strength, competing agents (includinginhibitors of binding) can be determined.

The differences in normal and abnormal cells can be detected using acomparison of EQELS spectra. For example, platelets with congenitalabnormalities, such as Glantzmann's thromboesthinia or theBenard-Soulier syndrome, bind to certain ligands abnormally, and thisabnormal binding may be detected in a EQELS spectrum before and afterthe ligand is added to a platelet medium. Leukemic blasts can also bedifferentiated from normal blasts by comparing a EQELS spectrum ofnormal blasts to an EQELS spectrum of Leukemic blasts. The production bythe host of an abnormal product, such as a monoclonal antibody or apolyclonal antibody, can also be detected.

Example 4 Drug Developments

The efficacy of various types of therapeutic agents on a microbe and/ora cell can also be assessed. An EQELS spectrum can be obtained from asample before and/or after a therapeutic agent is administered.Therapeutic agents that may be assessed in this manner include drugs,hormonal agents, leukemic therapeutics, anti-platelet effects,pharmacological agents, vitamins, pH conditions or analytes. The systemsand methods of the present invention may also be used to evaluate,adjust and/or identify therapies in drug/treatment development programsand/or in clinical or pre-clinical drug trials or other drug developmenttesting, including clinical or pre-clinical trials for developing orevaluating vitamin supplements, herbal remedies, and/or othertreatments. Embodiments of the present invention may also be used toevaluate, adjust and/or identify a suitable dose of a selected treatmentbased on the effectiveness of the treatment as measured by EQELSspectra. Patient specific assessments can be made to select appropriatetherapeutic agents. In some embodiments, no reporter label is required,and the process is non-destructive to either the cell under study or tothe ligand.

During the development of a drug, the chemical structure of the drug canbe refined. Thus, large numbers of drugs may be screened before theselection of the lead compound. This same process can be applied to thedevelopment of biologics. Embodiments of the invention can be used toevaluate the efficacy of various compounds, including peptides,proteins, lipids, nucleic acids, and/or small molecules. Examples ofinteractions that can be evaluated using one or more EQELS spectrainclude the binding of coagulation factors to activated platelets, theinhibition of platelet agonists, the selective binding to neoplastictissue compared to normal tissue, surface activation and/or enzymesinteraction. The detection of therapeutic agent and cell or microbeinteraction and the assessment of the biologic, chemical and/orthermodynamic character of the binding can be used to select promisingtherapeutic agents for specific uses.

Example 5 Detection of Recombinant Human Factor VIIa (rFVIIa) Activationof Factor FIX on Activated Platelets Using EQELS Spectra

1. Summary

FIX can be activated by FXIa or by FVIIa/TF in the presence of calcium.The role of FVIIa/TF in the activation of FIX appears to be theinitiating event, although activation of FIX by both mechanisms isimportant. In the presence of calcium, both FIX and FIXa bind to theactivated platelet surface with a Kd of 8 nM and 2 nM, respectively. Inthe presence of 3 nM rFVIIIa, the binding of FIX and FIXa to activatedplatelets is tighter with a Kd of 5.8 nM and 0.6 nM, respectively. AtrFVIIa concentrations less than 100 nM, no direct binding to theactivated platelet surface can be detected with light scattering even inthe presence of calcium. However, in the presence of FIX, rFVIIa bindsto platelets at concentrations as low as 10 nM rFVIIa. Furthermore,FVIIa on the surface of activated platelets can activate FIX in theabsence of tissue factor. This is reflected by an increase bindingaffinity and a decrease in the FIX Kd from 8 to 1.6 nM. When rFVIIa isadded to activated platelets in the presence of both FIX and FVIIIa, theKd for FIX decreases to 0.6, suggesting that rFVIIa activates FIX on thesurface of activated platelets in the absence of tissue factor. Theactivation of factor IX by FVIIa on activated platelets can also bedemonstrated by a functional assay for FIXa. These data suggest that theeffectiveness of pharmacologic doses of rFVIIa in bleeding patients,such as those sustaining severe blunt trauma may be, at least in part,due to the direct activation of FIX by rFVIIa to form additional tenasecomplexes ultimately resulting in improved thrombin generation. Thesereactions can occur even in the absence of tissue factor.

2. Introduction

The modern view of the initiation and assembly of coagulation activationcomplexes such as the tenase complex suggests that cellular surfaces areof fundamental importance for the formation and localization of thrombingeneration on the platelet surface resulting in a hemostatic plug. SeeMonroe, D. M. and Roberts, H. R. Mechanism of action of high-dose factorVIIa: points of agreement and disagreement. Arterioscler Thromb VascBiol. (2003) 23(1):8-9. Coagulation initiation involves cell-boundtissue factor (TF) and activated coagulation factor FVII (FVIIa). SeeRauch, U., Bonderman, D., Bohrmann, B., Badimon, J. J., Himber, J.,Riederer, M. A. and Nemerson Y. Transfer of tissue factor fromleukocytes to platelets is mediated by CD15 and tissue factor. Blood(2000) 96:170-5. The occupancy of the TF receptor by FVIIa may beimportant because the TF/FVIIa complex can activate both zymogen factorX (FX) for participation in the prothrombinase complex and zymogenfactor IX (FIX) for participation in the tenase complex: In the case ofeither congenital or acquired deficiencies or inhibition of componentsof the tenase or prothrombinase complex, either replacement of themissing factor or the use of agents thought to “by-pass” certaindeficiencies may restore normal hemostasis. There is now increasingevidence that the administration of pharmacologic doses of recombinanthuman FVIIa (rFVIIa) may act as a by-passing agent. Evidence to datesuggests that the “by-passing” action of rFVIIa is due to both theenhancement of the TF-pathway as well as the direct activation FX byrFVIIa on the surface of activated platelets. See Roberts and Monroe, D.M. Newer concepts of blood coagulation. Haemophilia. (1998) 4(4):331-4.

FIX activation may proceed through interaction with TF/FVIIa complex aswell as by FXIa, the latter occurring on the surface of activatedplatelets. Di Scipio, R., Kurachi, K., and Davie, E. W. (1978) J ClinInvest 61, 1528-1538. Fujikawa, K., Legaz, M., Kato, H., and Davie, E.The mechanism of activation of bovine factor IX (Christmas factor) bybovine factor XIa, activated plasma thromboplastin antecedent. (1974)Biochemistry 13, 4508-4516; Osterud, B., Bouma, B., and Griffin, J.Human blood coagulation factor IX. Purification, properties, andmechanism of activation by activated factor XI (1978) J Biol Chem 253,5946-5951; Kurachi, K., and Davie, E. W. Isolation and characterizationof a cDNA coding for human factor IX (1982) Proc Natl Acad Sci USA 79,6461-6464; Zur, M., and Nemerson, Y. Kinetics of factor IX activationvia the extrinsic pathway. Dependence of Km on tissue factor (1980) JBiol Chem 255, 5703-5707; Osterud, B., and Rapaport, S. Activation offactor IX by the reaction product of tissue factor and factor VII:additional pathway for initiating blood coagulation (1977) Proc NatlAcad Sci USA 74, 5260-5264; Hoffman, M., Monroe, D., and Roberts, H.Coagulation factor IXa binding to activated platelets andplatelet-derived microparticles: a flow cytometric study (1992) Thromb.Haemost. 68, 74-78; Hoffman, M., Pratt, C. N. Corbin, L. W., Church, F.C. Characteristics of the chemotactic activity of heparin cofactor IIproteolysis products (1990) J. Leukoc. Biol. 265, 156-162; Bauer, K.,Kass, B., ten Case, H., Hawinger, J., and Rosenberg, R. Factor IX isactivated in vivo by the tissue factor mechanism (1990) Blood 76,731-736. FIXa binds to the activated platelet surface with a Kd of 2.5nM, but in the presence of rFVIIIa, FIXa binding is tighter with a Kd of0.6 nM. See Osterud, B., Bouma, B., and Griffin, J. Human bloodcoagulation factor IX. Purification, properties, and mechanism ofactivation by activated factor XI (1978) J Biol Chem 253, 5946-5951;Ahmad, S., Rawala-Sheikh, R., and Walsh, P. Comparative interactions offactor IX and factor IXa with human platelets J Biol Chem. 1989 Feb. 25;264(6):3244-51; Ahmad, S., Rawala-Sheikh, R., Monroe, D., Roberts, H.,and Walsh, P. Comparative platelet binding and kinetic studies withnormal and variant factor IXa molecules (1990) J Biol Chem 265,20907-20911; Ahmad, S., Rawala-Sheikh, R., Cheug, W., Stafford, D., andWalsh, P. The role of the first growth factor domain of human factor IXain binding to platelets and in factor X activation (1992) J Biol Chem267, 8571-8576; Rawala-Sheikh, R., Ahmad, S. S., Ashby, B., Walsh, P. N.Kinetics of coagulation factor X activation by platelet-bound factor IXa(1990) Biochem. 29, 2606-2611; Rao, L. V., Rapaport, S. I. Activation offactor VII bound to tissue factor: a key early step in the tissue factorpathway of blood coagulation. (1988) Proc. Natl. Acad. Sci. (USA) 85,6687-6691. There are approximately 300 binding sites for FIX and 550 forFIXa.

It is widely accepted that complex formation between FVIIa and TF is anobligatory step for the expression of FVIIa activity. See Fujikawa, K.,Legaz, M., Kato, H., and Davie, E. The mechanism of activation of bovinefactor IX (Christmas factor) by bovine factor XIa, activated plasmathromboplastin antecedent (1974) Biochemistry 13, 4508-4516; Rao, L. V.,Rapaport, S. I. Activation of factor VII bound to tissue factor: a keyearly step in the tissue factor pathway of blood coagulation (1988)Proc. Natl. Acad Sci. (USA) 85, 6687-6691; Nemerson, Y., Gentry, R. Anordered addition, essential activation model of the tissue factorpathway of coagulation: evidence for a conformational cage (1986)Biochem. J. 25, 4020-4033; Lawson, J. H., Mann, K. G. Cooperativeactivation of human factor IX by the human extrinsic pathway of bloodcoagulation (1991) J. Biol. Chem. 266, 11317-11327. However, severalstudies have demonstrated the TF independent activation of FX by highdoses of FVIIa in the presence of calcium ion alone or calcium andphospholipid vesicles. See Bom, V., and Bertina, R. The contributions ofCa2+, phospholipids and tissue-factor apoprotein to the activation ofhuman blood-coagulation factor X by activated factor VII (1990) Biochem.J. 265, 327-336; Komiyama, Y., Pederson, A. H., Kisiel, W. Proteolyticactivation of human factors IX and X by recombinant human factor VIIa:effects of calcium, phospholipids, and tissue factor (1990) Biochem. J.29, 9418-9425; Miletich, J. P., Jackson, C. M., Majerus, P. W.Interaction of coagulation factor Xa with human platelets (1977) Proc.Natl. Acad. Sci. (USA) 74, 4033-4036; Hoffman, M., Monroe, D. M.,Oliver, J. A., and Roberts, H. R. Factors IXa and Xa play distinct rolesin tissue factor-dependent initiation of coagulation (1995) Blood 86,1794-1801.

By monitoring changes in the binding constant of FIX during itsactivation by rFVIIa, it can be shown that factor FIX can be convertedto FIXa. Unactivated FIX that is bound to activated platelets canprovide an interaction site for rFVIIa and the rFVIIa-FIX interactioncan lead to activation of FIX. Once FVIIa binds to and activates FIX,the newly formed FIXa in the presence of FVIIIa can be assembled intoadditional tenase complexes, thus, enhancing thrombin production.

3. Experimental Procedures

Platelet isolation. Fresh gel-filtered platelets were used in allexperiments. Blood from healthy donors was collected inacid-citrate-dextrose and prostacyclin (5 Φg/mL, PGI₂, Sigma ChemicalCo., St. Louis, Mo.). Platelet-rich-plasma was obtained bycentrifugation of the anticoagulated blood at 150 g for 15 minutes. Theplatelets were isolated from PRP by centrifugation at 650 g for 20minutes, and resuspended in citrated saline (13 mM citrate, 123 mM NaCl,50 mM dextrose) buffer containing 5 Φg/mL PGI₂, washed twice and thenresuspended in a small amount of calcium-free albumin-free Tyrode'sbuffer containing 5 Φg/mL prostacyclin (PGI₂). Gel-filtered plateletswere obtained from the application of washed platelets to a SepharoseCL-2B column (Pharamica Inc., Uppsala, Sweden) equilibrated withcalcium-free albumin-free Tyrode's buffer. Platelets for lightscattering experiments were suspended in buffer containing 20 mM NaCl,265 mM sucrose, 2 mM HEPES, pH 7.4 and activated with human ∀-thrombin(0.2 NIH units/mL). See Li, X., Gabriel, D. A. The physical exchange offactor VIII (FVIII) between von Willebrand factor and activatedplatelets and the effect of the FVIII B-domain on platelet binding(1997) Biochem. J. 36, 10760-10767. Fresh gel filtered platelets wereshown to activate and aggregate normally with 1 U/ml of human thrombinand 10 μM ADP. EQELS spectra of the gel filtered platelets gave a singlehomogeneous mobility indicating the lack of microparticles. No change isobserved in the platelet mobility spectrum on the addition of rFVIIaover the concentration range where rFVIIa would be expected to bind totissue factor if it was present, indicating an absence of TF in thepreparation.

Proteins. Blood coagulation FIX and FIXa were purified. See McCord, D.M., Monroe, D. M., Smith, K. J., Roberts, H. R. Characterization of thefunctional defect in factor IX Alabama. Evidence for a conformationalchange due to high affinity calcium binding in the first epidermalgrowth factor domain. (1990) J. Biol. Chem. 265, 10254-10259.Recombinant human FVIIa (rFVIIa) was provided by Dr. Ulla Hedner of NovoNordisk, Copenhagen, Denmark. Recombinant human Factor VIII (rFVIII) wasprovided by Bayer Laboratories Inc., Clayton, N.C.

Electrophoretic Quasi-Elastic Light Scattering (EQELS). Electrophoreticquasi-elastic light scattering offers the ability to monitor changes inthe surface of blood cells resulting from cell activation and ligandbinding. Gabriel, D. A., Reece, N., Witte, J., and Muga, K.Electrophoretic light scattering studies on the interaction offibrinogen with resting and activated human platelets (1993) BloodCoagul. Fibrinolysis 4, 397-403; Johnson, C., and Gabriel, D. A. (1995)Laser Light Scattering, Dover, N.Y. The electrophoretic mobility ofactivated platelets changes when exposed to a known ligand and that themobility change is the result of ligand binding. It is the change in theplatelet surface charge density and hence its electrophoretic mobilitycaused by ligand binding and not the ligand itself that is monitored.Under these experimental conditions, resting gel-filtered platelets havean electrophoretic mobility of −0.9 (Φ-cm)/(volt-sec) and activatedplatelets have a mobility of −0.65(Φ-cm)/(volt-sec). Loading theplatelet surface with a ligand, such as a FIX or FVIII, changes theplatelet electrophoretic mobility. While the absolute mobility variesslightly from donor to donor, there is minimal variation in a givendonor. The dependence of the change in the platelet electrophoreticmobility on the addition of FIX permits calculation of the bindingconstant for FIX to the activated platelet. It is not the magnitude ofthe change in the platelet electrophoretic mobility, Φ, that determinesthe binding constant, but how the change in μ depends on theconcentration of the ligand FIX. The effect of ligand binding on theplatelet mobility varies with the ligand, and is dependent on the extentof ligand binding, the extent of surface modification caused by ligandbinding, and on the net charge of the ligand itself.

EQELS measurements were made on a multi-angle quasi-elastic lightscattering spectrometer (DELSA 440, Coulter Electronics, Inc., Hialeah,Fla.). See Li, X., Gabriel, D. A. Biochem. J. 36, 10760-10767 (1997);Johnson, C., and Gabriel, D. A. (1995) Laser Light Scattering, Dover,N.Y.

Debye limit. Charged particles in medium orient oppositely chargedcounter ions about their surface so that the electrical potential of theparticle's surface decreases with the distance from the particlesurface. At a distance defined by the Debye-Huckel theory, the particleno longer has an influence on the medium counter ions. The distance fromthis point to the particle surface is called the Debye-Huckel length,also called the electrical double layer, and for platelets is estimatedto be 8Δ. See Jung, S., Kinoshita, K., Tanoue, K., Isohisa, I., andYamazaki, H. Role of surface negative charge in platelet functionrelated to the hyperreactive state in estrogen-treated prostaticcarcinoma (1982) Thromb Haemost 47, 203-209; Pethica, B. (1961) Exp CellRes 8, 123-140. The electrophoretic mobility for particles the size ofblood cells, where the ratio of the particle diameter to the Debyescreening length is greater than 30, assuming a platelet diameter of atleast 10,000Δ, is governed by the magnitude of the surface chargedensity and not by frictional factors. See Ware, B. (1974) AdvancedColloid Interface Science 4, 1-44. Smoluchowski, M. (1921) Handbuch derElektrizitat und des Magnitisums. (Barth, L., Ed.)

Binding of proteins to platelets. Activated platelets and activatedFVIII were prepared by activation with 0.2 NIH U/mL human ∀-thrombin for5 minutes. After activation, residual thrombin was inactivated byaddition of 2.5 ΦM phenylalanyl prolinyl arginine chloromethyl ketone(PPACK, Calbiochem, La Jolla, Calif.). FIX, FIXa, FVIIIa, and rFVIIawere incubated with platelets for 10 minutes before EQELS measurements.

The binding coefficient (K_(d)) of the proteins to platelets wasdetermined by fitting the data from the binding experiment to:

$\mu = {\mu_{0} + {\Delta\;\mu\frac{\lbrack{Ligand}\rbrack}{K_{d} + \lbrack{Ligand}\rbrack}}}$where, μ is the electrophoretic mobility, μ₀ is the mobility in theabsence of added protein, Δμ is the calculated maximal change inmobility at the saturating concentration of protein. This model assumesone class of binding sites for the specific protein ligand on theplatelets. Data was fitted using the nonlinear module (NLIN) of theanalysis program SAS (SAS Institute Inc., Cary, N.C.).

Zymogen FIX at varying concentrations was added to the thrombinactivated platelets that in some experiments included FVIIIa followed bya 10 minute incubation period. After this incubation period, rFVIIa wasadded to activated platelets coated with zymogen FIX. The EQELS spectrumwas then obtained.

Functional assays for activation of FIX and FX. All assays were run inTyrodes buffer with 2 mM CaCl₂. Platelets were activated with 0.2 NIHunits/ml of human α-thrombin for 10 minutes at 37° C. FVIII at 5units/ml were included with the platelets and thrombin. Thrombin wasneutralized with hirudin from a medium titrated against an α-thrombinstandard. FVIIa at varying concentrations was added to the platelets for10 minutes. Plasma concentrations of FIX at 80 nM were added andincubated for varied times. FIXa was assayed in a two stage assay.Platelets with activated FIX were incubated with plasma concentrationsof FX for 5 minutes, then the amount of FXa was measured by addingplasma concentrations of prothrombin along with a prothrombin substrate(0.5 mM Perfachrome Th). Pilot studies showed that: FXa generation waslinear over the 5 minute assay period, that FXa generation was dependenton FIX, that thrombin generation was dependent on the addition of FX,and that there was no background cleavage of substrate in the absence ofprothrombin. Thrombin generation was measured as the change in theabsorbance as the p-nitroanilide substrate was cleaved as a function oftime. As expected, substrate cleavage fit a second order pattern and therate of thrombin generation was determined from the first derivative ofthe absorbance versus time data.

4. Results

In FIG. 8 the binding profile for zymogen FIX interaction with activatedplatelets in the presence of 4 mM calcium and in the presence andabsence of rFVIIIa is shown. In the absence of rFVIIIa, unactivated FIXbinds with a Kd of 7.9 nM (CI=6.948 to 8.851). When 3 nM FVIIIa ispresent, the binding of FIX to activated platelets is slightly tighteras seen by a decrease in the Kd to 5.8 nM (CI=5.183 to 6.419). TheseKd's are different based on comparison of non-overlapping 95% confidenceintervals.

FIG. 9 depicts the binding of activated FIX to activated platelets. Incontrast to activated platelets, resting platelets do not bind eitherFIX or FIXa (data not shown). In the presence of 4 mM calcium ion, butno rFVIIIa, FIXa binds to activated platelets with a Kd of 1.9 nM(SE=0.05, CI=1.741 to 2.059). See Roberts and Monroe, D. M. Newerconcepts of blood coagulation Haemophilia. (1998) 4(4):331-4; Di Scipio,R., Kurachi, K., and Davie, E. W. (1978) J Clin Invest 61, 1528-1538;Fujikawa, K., Legaz, M., Kato, H., and Davie, E. The mechanism ofactivation of bovine factor IX (Christmas factor) by bovine factor XIa,activated plasma thromboplastin antecedent (1974) Biochemistry 13,4508-4516; Osterud, B., Bouma, B., and Griffin, J. Human bloodcoagulation factor IX. Purification, properties, and mechanism ofactivation by activated factor XI (1978) J Biol Chem 253, 5946-5951;Kurachi, K., and Davie, E. W. Isolation and characterization of a cDNAcoding for human factor IX (1982) Proc Natl Acad Sci USA 79, 6461-6464;Hoffman, M., Monroe, D., and Roberts, H. Coagulation factor IXa bindingto activated platelets and platelet-derived microparticles: a flowcytometric study (1992) Thromb. Haemost. 68, 74-78; Ahmad, S.,Rawala-Sheikh, R., and Walsh, P. Comparative interactions of factor IXand factor IXa with human platelets J Biol Chem. 1989 Feb. 5;264(6):3244-51; Ahmad, S., Rawala-Sheikh, R., Monroe, D., Roberts, H.,and Walsh, P. Comparative platelet binding and kinetic studies withnormal and variant factor IXa molecules (1990) J Biol Chem 265,20907-20911; Rao, L. V., Rapaport, S. I. Activation of factor VII boundto tissue factor: a key early step in the tissue factor pathway of bloodcoagulation (1988) Proc. Natl. Acad Sci. (USA) 85, 6687-6691; Lawson, J.H., Mann, K. G. Cooperative activation of human factor IX by the humanextrinsic pathway of blood coagulation (1991) J. Biol. Chem. 266,11317-11327; Bom, V., and Bertina, R. The contributions of Ca2+,phospholipids and tissue-factor apoprotein to the activation of humanblood-coagulation factor X by activated factor VII (1990) Biochem. J.265, 327-336. However, as shown in FIG. 9, in the presence of 4 mMcalcium and 3 nM rFVIIIa, the Kd for FIXa binding to activated plateletsdecreases from 1.9 nM to 0.569 nM (SE=0.185, CI=0.188 to 0.950) showingtighter binding. The confidence intervals of the Kd values for FIXabinding with and without rFVIIIa are statistically different at α=0.05.Because of its significant charge, rFVIIIa confers a higher surfacecharge to the activated platelets, observed by the downward shift of thebinding curve when rFVIIIa is present. Li, X., Gabriel, D. A. Biochem.J. 36, 10760-10767 (1997).

Because of the difference in the binding constant between FIX and FIXaand since rFVIIa does not bind to activated platelets at 10 nM, thefollowing experiments were performed to investigate the role of rFVIIain the activation of platelet bound zymogen FIX. A decrease in the Kd ofFIX was used to detect activation of zymogen FIX by rFVIIa.

FIG. 10 shows the effect of a high concentration of rFVIIa on theactivation of zymogen FIX. In these experiments 10 nM rFVIIa and avariable amount of zymogen FIX (0 to 20 nM) was added to thrombinactivated platelets and incubated for 10 minutes followed by thedetermination of the electrophoretic mobility. When rFVIIa is present ata concentration of 10 nM, FIX binds to activated platelets with a Kdobserved for FIXa in the absence of rFVIIIa (Kd=1.62, CI=1.491 to 1.748)compared to a Kd of 1.9 for FIXa shown in FIG. 9. When 3 nM rFVIIIa isfirst added to the activated platelets, incubated for 10 minutes,followed by addition of zymogen FIX and rFVIIa and reincubated for 10minutes, the binding is tighter (Kd=0.84, CI=0.287 to 1.393). Thedifference between these binding curves is also statisticallysignificantly different. The tighter binding is reflected in thedecrease in the Kd and is similar to that observed for FIXa binding toplatelets in the presence of rFVIIIa (compare to FIG. 9 and Table I).These experiments suggest that at pharmacologic doses of rFVIIa, zymogenFIX is activated by rFVIIa on activated platelets in the absence of TF.

TABLE I Summary of Binding Coefficients for FIX and FIXa Interactionwith Activated Platelets. Conditions FIX FIXa FIX + FVIIa* No Ca⁺⁺, NoFVIIIa — — — 4 mM Ca⁺⁺, No FVIIIa 7.9 (CI 6.95-8.85)  1.9 (CI 1.74-2.06) 1.6 (CI 1.49-1.750) 4 mM Ca⁺⁺, 3 nM FVIIIa 5.8 (CI 5.18-6.42) 0.57 (CI0.19-0.95) 0.84 (CI 0.29-1.39) *The Kd for FIX in the presence of FVIIais very similar to that for direct activation of FIX and indicates thatFIX is activated by FVIIa on the activated platelet surface.

In FIG. 11A the time dependence for the activation of zymogen FIX (10nM) at different concentrations of rFVIIa is shown. The rate constantsfor these reactions are shown in FIG. 11B. At 0.075 nM rFVIIa minimalactivation of zymogen FIX is observed at approximately 30 minutes (FIG.11A and the first data point in FIG. 11B). When the concentration ofrFVIIa is increased to 1 nM, further activation of zymogen FIX isdetected. When the concentration of rFVIIa is further increased to 10nM, the conversion of zymogen FIX to activated FIX occurs at a stillmore rapid rate.

Tissue factor was not present on the surface of activated platelets inthe experiments presented. The evidence for this is that addition ofrFVIIa over a concentration range of 0 to 40 nM did not change theelectrophoretic mobility of platelets. If tissue factor were present,rFVIIa would bind to tissue factor and induce an alteration in theplatelet surface charge that would be observed as a change in theplatelet mobility.

FIG. 12 provides additional functional evidence for the activation ofplatelet-bound zymogen FIX by rFVIIa. In the absence of rFVIIa zymogenFIX is not activated (filled circles FIG. 12). As rFVIIa is added at 10nM (squares), 20 nM (diamonds), 30 nM (inverted triangles), and 40 nM(triangles), the activation of zymogen FIX is shown to be linear in timeand directly proportional to the concentration of added rFVIIa. The rateconstant for FIX activation as a function of the concentration of addedrFVIIa is shown in the inset of FIG. 12. Although not reflected in FIG.12, it has also been shown that FXa and thrombin are produced in thissystem and also dependent on the concentration of added rFVIIa. Datashown in FIG. 12 confirms the EQELS result that platelet-bound FIX isactivated by rFVIIa.

5. Discussion

A complete understanding of hemostasis, including contributions fromplatelets, soluble phase coagulation factors, surface effects and fluiddynamics, have been hindered by the complexity of the system. A recenttheory for coagulation proposed by Nemerson and others supports TF asthe initiating event in hemostasis. See Rauch, U., Bonderman, D.,Bohrmann, B., Badimon, J. J., Himber, J., Riederer, M. A. and NemersonY. Transfer of tissue factor from leukocytes to platelets is mediated byCD15 and tissue factor Blood (2000) 96:170-5; Nemerson, Y. Tissue factorand hemostasis (1988) Blood 71, 1-8; Rapaport, S. The extrinsic pathwayinhibitor: a regulator of tissue factor-dependent blood coagulation(1991) Thromb Haemost 66, 6-15; Rao, L. V., and Rapaport, S. Theextrinsic pathway inhibitor: a regulator of tissue factor-dependentblood coagulation (1990) Blood 75, 1069-1073; Broze, G. The role oftissue factor pathway inhibitor in a revised coagulation cascade (1992)Sem Hematol 29, 159-169. Physical separation of the amplifying tenaseand prothrombinase complexes from the TF/FVIIa trigger complex providesa spatial feature for regulation of hemostasis. It seems likely thatsoluble zymogen FIX is first activated by the TF-FVIIa complex at a sitedistant to the platelet surface and then translocation of activated FIXto the platelet surface. The activated platelet participates in thissequence through tight binding of coagulation factors to specificbinding sites, so that amplification complexes can be sequestered,spatially oriented, and protected from plasma inhibitors.

In the special case where high concentrations of rFVIIa are infused asin the treatment of hemophilic patients or for bleeding episodes innon-hemophiliacs, the circumstances for FIX activation may be different.In this case rFVIIa may be bound to other sites in addition to TF. Inthis scheme, zymogen FIX bound to the activated platelet surface couldprovide a binding site for rFVIIa, since rFVIIa at physiologicconcentrations does not directly bind to the platelet surface itself. Itcan be shown that at very high concentrations, rFVIIa will bind toplatelets in the absence of calcium with a Kd of 250 nM and in thepresence of calcium with a Kd of 123 nM (unpublished data). It isestimated that the concentration of rFVIIa after therapeutic infusionfor a bleeding diathesis is approximately 30 nM, borderline for bindingdirectly to the activated platelet surface. Under these conditionsrFVIIa may bind directly to zymogen FIX bound to the platelet surface.Monroe and colleagues have previously reported evidence that rFVIIabound to activated platelets can directly activate FX. See Nelsestuen GL, Stone M, Martinez M B, Harvey S B, Foster D, Kisiel W. Elevatedfunction of blood clotting factor VIIa mutants that have enhancedaffinity for membranes: Behavior in a diffusion-limited reaction J BiolChem. 2001 Oct. 26; 276(43):39825-31.

Platelets are intimately involved at several steps of the coagulationpathway. It was found that activated platelets promote the activation ofFX to form FXa by a complex of FIXa, FVIIIa, and calcium. Ahmad, S.,Rawala-Sheikh, R., and Walsh, P. Platelet receptor occupancy with factorIXa promotes factor X activation (1989) J Biol Chem 264, 20012-20016;Hultin, M. Role of human factor VIII in factor X activation (1982) JClin Invest 69, 950-958; van Rijn, J., Rosing, J., and van Dieijen, G.Activity of human blood platelets in prothrombin and in factor Xactivation induced by ionophore A23187. (1983) Eur. J Biochem. 133,1-10; Neuenschawander, P., and Jesty, J. A comparison of phospholipidand platelets in the activation of human factor VIII by thrombin andfactor Xa, and in the activation of factor X (1988) Blood 72, 171-177.Activated platelets not only provide the phospholipid surface, but alsopresumably possess specific, high affinity, saturable binding sites forFXa (See Miletich, J. P., Majerus, D. W. and Majerus, P. W. A comparisonof phospholipid and platelets in the activation of human factor VIII bythrombin and factor Xa, and in the activation of factor X (1978) J.Clin. Invest. 62, 824-831; Tracy, P., Nesheim, M., and Mann, K.Coordinate binding of factor Va and factor Xa to the unstimulatedplatelet J Biol. Chem. 1981 Jan. 25; 256(2):743-51.), FVa (See Tracy,P., Nesheim, M., and Mann, K. Coordinate binding of factor Va and factorXa to the unstimulated platelet J Biol. Chem. 1981 Jan. 25;256(2):743-51; Tracy, P., Peterson, J., Weisheim, M., McDuffie, F. andMann, K. Interaction of coagulation factor V and factor Va withplatelet. (1979) J Biol. Chem. 254, 10354-10361), FVIIIa (See Li, X.,Gabriel, D. A. Biochem. J. 36, 10760-10767 (1997), Nesheim, M., Pittman,D., Way, J., Slonosky, D., Giles, A. and Kaufman, R. The binding of35S-labeled recombinant factor VIII to activated and unactivated humanplatelets (1988) J. Biol. Chem. 263, 16467-16470), and FIXa (See Ahmad,S., Rawala-Sheikh, R., and Walsh, P. Platelet receptor occupancy withfactor IXa promotes factor X activation (1989) J Biol Chem 264,20012-20016). TF-bearing microparticles possible derived from leukocytesappear to be significant to the thrombus perpetuation, but not in theinitiation of the thrombus. See Rauch, U., Bonderman, D., Bohrmann, B.,Badimon, J. J., Himber, J., Riederer, M. A. and Nemerson Y. Transfer oftissue factor from leukocytes to platelets is mediated by CD15 andtissue factor Blood (2000) 96:170-5. Microparticles containing TF thatlocalize to activated platelets that normally do not contain TF has beenshown to be mediated by P-selectin on the platelet surface and CD15 onthe microparticle. See Rauch, U., Bonderman, D., Bohrmann, B., Badimon,J. J., Himber, J., Riederer, M. A. and Nemerson Y. Transfer of tissuefactor from leukocytes to platelets is mediated by CD15 and tissuefactor Blood (2000) 96:170-5; Giesen, P. L., Rauch, U., Bohrmann, B.,Kling, D., Roque, M., Fallon, J. T., Badimon, J. J, Himber, J, Riederer,M. A. and Nemerson, Y. Blood-borne tissue factor: another view ofthrombosis. Proc Natl Acad Sci USA. (1999) 96:2311-2315; Halvorsen, H.,Olsen, J. O. and Osterud, B. Granulocytes enhance LPS-induced tissuefactor activity in monocytes via an interaction with platelets. J LeukocBiol. (1993) 54:275-82; Huge, B, Socie, G., Vu, T., Toti, F., Gluckman,E., Freyssinet, J. M. and Scrobohaci, M. L. Elevated levels ofcirculating procoagulant microparticles in patients with paroxysmalnocturnal hemoglobinuria and aplastic anemia Blood. (1999) 93:3451-6;Kirchhofer, D., Riederer, M. A. and Baumgartner, H. R. Specificaccumulation of circulating monocytes and polymorphonuclear leukocyteson platelet thrombi in a vascular injury model Blood. (1997) 89:1270-8.

The changes observed in the platelet surface charge as the concentrationof FIXa is increased (FIG. 9) are due to binding of FIXa to the plateletsurface since: 1) the binding is saturable, 2) the change requires thepresence of calcium, 3) platelet activation is required, 4) saturationwith other proteins does not inhibit the effect, and 5) a protein highlyhomologous to Factor IXa, i.e., FVIIa, does not change the plateletsurface charge under identical conditions. At concentrations of rFVIIagreater than 100 nM the binding of rFVIIa to activated platelets can bedetected. The possible role of TF-containing microparticles as a sourcefor this effect under these conditions is unlikely since a mobilitycorresponding to microparticles in the mobility spectrum is notobserved. Additionally, no change in the platelet mobility occurs whenrFVIIa is added at concentrations that should bind TF. Further supportfor specific FIXa binding is appreciated from the increased affinity ofactivated platelets for FIX in the presence of rFVIIIa (1.9 nM to 0.569nM). Ahmad, S., Rawala-Sheikh, R., and Walsh, P. Platelet receptoroccupancy with factor IXa promotes factor X activation (1989) J BiolChem 264, 20012-20016.

Previous studies on platelet activation monitored by EQELS showed thatplatelet activation results in a reduction in the surface chargeprobably due to the exposure of specific binding sites and theiroccupancy by specific ligands. Li, X., Gabriel, D. A. Biochem. J. 36,10760-10767 (1997). Gabriel, D. A., Reece, N., Witte, J., and Muga, K.Blood Coagul. Fibrinolysis 4, 397-403 (1993). The exact mechanism forthe platelet surface charge modification during platelet activation isnot known. It is known that many surface events occur with plateletactivation, e.g., exposure of CD62, FVa, various receptors, appearanceof phosphatidyl serine, etc., all of which could contribute to changesin the platelet surface charge. Electrophoretic light scattering canmonitor these changes. After platelet activation is complete, thesurface charge on the platelet surface stabilizes until a ligand isbound to the activated surface. Electrophoretic light scattering is alsohighly sensitive in monitoring the ligand-activated platelet surfaceinteraction.

One important result of these experiments is that high concentrations ofrFVIIa can activate FIX even in the absence of tissue factor, which mayexplain its effect in the correction of bleeding in non-hemophiliacs whohave a severe hemorrhagic diathesis.

The foregoing embodiments are illustrative of the present invention andare not to be construed as limiting thereof. The invention is defined bythe following claims, with equivalents of the claims to be includedtherein.

1. A system for identifying an unknown biological particle in a samplemedium, said system comprising: an Electrophoretic Quasi-elastic LightScattering (EQELS) spectrometer comprising an EQELS controllerconfigured to generate a EQELS spectrum for the biological particle inthe sample medium, wherein said biological particle comprises abiological cell; an EQELS analyzer in communication with said EQELSspectrometer; a reference database in communication with said EQELSanalyzer, said reference database comprising a plurality of spectra,each of said plurality of spectra corresponding to an EQELS spectrum forone of a plurality of different types of known biological cells; whereinsaid EQELS analyzer is configured to compare said EQELS spectrum for theunknown biological particle in the sample medium from said EQELSspectrometer with said plurality of spectra from said reference databaseand to identify the unknown biological particle in the sample mediumfrom the comparison.
 2. The system of claim 1, wherein said biologicalcell is a microbe selected from the group consisting of viruses,bacteria, fungi, and protozoa.
 3. The system of claim 1, wherein saidEQELS spectrum comprises a first EQELS spectrum, and said EQELSspectrometer further comprises: a sample chamber configured to receivethe sample medium; a sample modification system in communication withsaid sample chamber and configured to modify the sample; and whereinsaid EQELS spectrometer controller is configured to modify the samplewith said sample modification system after detecting said first EQELSspectrum and to detect a second EQELS spectrum after modifying thesample; wherein said EQELS analyzer is configured to compare said firstEQELS spectrum to said second EQELS spectrum to characterize thebiological particle in the sample medium from the comparison.
 4. Thesystem of claim 3, wherein said sample modification system is configuredto add an antibody to said sample medium, wherein the antibody is aspecific binder to a predetermined biological particle, and wherein saidEQELS analyzer is configured to determine if the biological particle inthe sample medium is the predetermined biological particle from thecomparison of said first EQELS spectrum to said second EQELS spectrum.5. The system of claim 3, wherein said modification system is configuredto modify the sample medium by adding a therapeutic agent to said samplemedium, and wherein said EQELS analyzer is configured to assess aneffectiveness of said therapeutic agent based on said comparison of saidfirst EQELS spectrum and said second EQELS spectrum.
 6. The system ofclaim 5, wherein said EQELS analyzer is configured to determine if thetherapeutic agent binds to a surface of the biological particle.
 7. Thesystem of claim 5, wherein the biological particle is a microbe and saidEQELS analyzer is configured to determine a change in swim rate of thebiological particle.
 8. The system of claim 5, wherein said EQELSanalyzer is configured to determine a binding constant for thetherapeutic agent.
 9. The system of claim 5, wherein the biologicalparticle is a microbe and said EQELS analyzer is configured to determinea swim rate for the microbe and to identify the microbe based on theswim rate, wherein said database includes swim rates for the pluralityof known microbes.
 10. The system of claim 1, wherein said EQELSspectrometer further comprises: an electric field generator for exposingthe biological particle in the sample medium to an electric field; alight source configured to impinge an excitation light on the biologicalparticle in the sample medium to produce scattered light; a detectorconfigured to detect the scattered light; wherein said EQELS analyzer isconfigured to detect a Doppler shift in the scattered light compared tothe excitation light.
 11. The system of claim 1, wherein the biologicalparticle is a microbe and wherein said EQELS analyzer is configured toidentify the microbe by determining a ratio of a swim rate of themicrobe to the electrophoretic mobility of the microbe.
 12. The systemof claim 1, further comprising a filtration device configured to collectthe biological particle from a fluid.
 13. The system of claim 12,wherein said filtration device further comprises: a filter configured tofilter a gas and/or liquid and to trap the biological particle therein;and a flushing inlet configured to flush the biological particle with afluid to provide the sample medium.
 14. The system of claim 13, whereinsaid filtration device is configured to collect a sample automatically.15. The system of claim 1, wherein the cell is a virus.
 16. The systemof claim 1, wherein the cell is a bacteria.
 17. The system of claim 1,wherein the cell is a fungi.
 18. The system of claim 1, wherein the cellis a protozoa.
 19. The system of claim 1, wherein the cell is a bloodcell.
 20. The system of claim 1, wherein the cell is a white blood cell.21. The system of claim 1, wherein the cell is a red blood cell.
 22. Thesystem of claim 1, wherein the cell is a platelet.
 23. The system ofclaim 1, wherein the cell is a cultured cell.
 24. The system of claim 1,wherein the cell is a suspended endothelial cell.
 25. The system ofclaim 1, wherein the cell is a nucleated cell.
 26. The system of claim1, wherein the cell is a non-nucleated cell.
 27. The system of claim 1,wherein the cell is a human cell.
 28. The system of claim 1, wherein thecell is a cloned cell.
 29. The system of claim 1, wherein the cell is aplant cell.
 30. The system of claim 1, wherein the cell is an animalcell.
 31. The system of claim 1, wherein the cell is a biopsied cell.32. The system of claim 1, wherein the cell is fixed with apreservative.